Vol. 282, Issue 2, R492-R500, February 2002
Involvement of trigeminal spinal nucleus in parasympathetic
reflex vasodilatation in cat lower lip
Kentaro
Mizuta1,2,
Satoshi
Kuchiiwa3,
Takashi
Saito2,
Hideaki
Mayanagi2,
Keishiro
Karita1, and
Hiroshi
Izumi1
1 Departments of Oral Molecular Bioregulation and
2 Pediatric Dentistry, Tohoku University Graduate School of
Dentistry, Sendai 980-8575; and 3 Department of Anatomy,
Faculty of Medicine, Kagoshima University, Kagoshima 890-8520, Japan
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ABSTRACT |
We examined
whether the trigeminal spinal nucleus (Vsp) forms part of the central
mechanism by which electrical stimulation of the central cut end of the
lingual nerve (LN) evokes parasympathetic reflex vasodilatation in the
lower lip in artificially ventilated, cervically vagosympathectomized
cats deeply anesthetized with 
-chloralose and urethane. For this
purpose, we made microinjections within the brain stem to produce
nonselective, reversible local anesthesia (lidocaine) or
soma-selective, irreversible neurotoxic damage (kainic acid). Local
anesthesia of Vsp by microinjection of lidocaine (2%; 1 µl/site)
reversibly and significantly reduced the ipsilateral-LN-evoked
parasympathetic reflex vasodilatation. Unilateral microinjection of
kainic acid (10 mM/site; 1 µl) into Vsp ipsilateral to the stimulated
LN led to an irreversible reduction in the reflex vasodilatation but
had no effect on the vasodilatation elicited by stimulation of the
contralateral LN. Such microinjection of kainic acid into Vsp had no
effect on the vasodilatation evoked by electrical stimulation of the
ipsilateral inferior salivatory nucleus. Electrical stimulation of Vsp
elicited a blood flow increase in the lower lip in an intensity- and
frequency-dependent manner, regardless of whether systemic arterial
blood pressure rose or fell. Hexamethonium (1.0 mg/kg iv) significantly
reduced the vasodilator responses elicited by electrical stimulation of
the central cut end of LN or of Vsp, each to a similar degree. After
hexamethonium, both vasodilator responses showed time-dependent
recovery. These results strongly suggest that Vsp is an important
bulbar relay for LN-evoked parasympathetic reflex vasodilatation in the
cat lower lip.
autonomic reflex; lidocaine; kainic acid; autonomic ganglion
blocker
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INTRODUCTION |
ELECTRICAL
STIMULATION of the central cut end of a branch of the trigeminal
nerve, such as the lingual nerve (LN), inferior alveolar nerve (IAN),
or infraorbital nerve, elicits a variety of responses mediated via
parasympathetic reflex mechanisms. These include vasodilatation in the
orofacial area and salivary or lacrimal secretions from the
submandibular gland, parotid gland, and lacrimal gland (18, 21,
39, 41). Although the peripheral neural pathways (afferent and
efferent nerves) involved in these reflex arcs have been clarified over
the last decade (21, 23, 24, 26, 29), the central
(medullary) neural circuitry remains undefined.
When electrically stimulated, LN and IAN elicit similar vasodilator
responses in the orofacial area (16, 19, 23) and both send
fibers to the trigeminal spinal nucleus (Vsp) (4, 37).
This nucleus is known to be a brain stem relay for nociceptive inputs
from the orofacial and visceral areas, which travel via the trigeminal,
glossopharyngeal, and vagus nerves (4). This may suggest
that trigeminal stimulation (nociceptive stimulation) is more likely to
be involved in evoking such parasympathetic reflex vasodilatation than
gustatory stimulation. This led us to examine the contribution made by
Vsp to the parasympathetic reflex vasodilatation in the cat lower lip
elicited by centrally directed stimulation of LN. For this purpose, we
made microinjections within the brain stem to produce nonselective,
reversible local anesthesia (lidocaine) or soma-selective, irreversible
neurotoxic damage (kainic acid). We also examined whether electrical
stimulation of Vsp elicits a similar parasympathetic vasodilatation.
The vascular bed of the cat's lower lip was selected for these
experiments because the peripheral neural pathways followed by the
parasympathetic vasodilator fibers have been well defined [for review,
see Izumi (14)].
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METHODS |
Preparation of animals.
The experimental protocols were reviewed by the Committee on the Ethics
of Animal Experiments in Tohoku University School of Medicine, and they
were carried out in accordance with both the Guidelines for Animal
Experiments issued by the Tohoku University School of Medicine and The
Law (No. 105) and Notification (No. 6) issued by the Japanese Government.
Thirty-one adult cats, unselected as to sex and of 2.0-4.5 kg body
wt (approximate age 2-4 yr), were initially sedated with ketamine
hydrochloride (30 mg/kg im) and then anesthetized with a mixture of
-chloralose (50 mg/kg iv) and urethane (100 mg/kg iv). These
anesthetics were supplemented if and when necessary throughout the
experiment (see below). Local anesthesia (2% lidocaine; 1-2 ml)
was applied to all skin incisions. A femoral artery was cannulated for
the measurement of systemic arterial blood pressure (SABP). One
cephalic vein was cannulated to allow drug injection. The anesthetized
animals were intubated, paralyzed by intravenous injection of
pancuronium bromide (Mioblock; Organon, Teknika, Netherlands; 0.4 mg/kg
initially, supplemented with 0.2 mg/kg every hour or so after testing
the level of anesthesia; see below) and artificially ventilated via the
tracheal cannula with a mixture of 50% air-50% O2.
The ventilator (model SN-480-6; Shinano, Tokyo, Japan) was set to
deliver a tidal volume of 10-12 cm3/kg at a rate of 20 breaths/min, and the end-tidal concentration of CO2 was
determined by means of an infrared analyzer (Capnomac Ultima; Datex,
Helsinki, Finland) as reported previously (13, 16, 22).
End-tidal CO2 was kept at 35-40 mmHg. Ringer solution (Otsuka Pharmaceutical, Tokyo, Japan) was continuously infused at a
rate of ~5 ml/h. Rectal temperature was maintained at 37-38°C using a heating pad.
In all experiments, the cervical vagi and superior cervical sympathetic
trunks were cut bilaterally in the neck before any stimulation to
eliminate the reflex actions of the vagus nerve on the cardiovascular
system and the effects of sympathetic vasoconstrictor fibers on the
orofacial area, respectively.
The criterion for the maintenance of an adequate depth of anesthesia
was the absence of a reflex elevation of SABP in response to a noxious
stimulus (such as pinching the upper lip for ~2 s). If the depth of
anesthesia was considered inadequate, additional -chloralose and
urethane (i.e., intermittent doses of 5 and 10 mg/kg iv, respectively)
were administered. Once an adequate depth of anesthesia had been
attained, supplementary doses of pancuronium were given approximately
every 60 min to maintain immobilization during periods of stimulation.
Electrical stimulation of LN.
To elicit a parasympathetic reflex vasodilatation in the lower
lip, the central cut end of LN was electrically stimulated (Fig.
1). The routine stimulus parameters were
a 20-s train of 2-ms rectangular pulses at a frequency of 10 Hz and at
supramaximal intensity (usually 30 V), as described previously
(13, 16, 22). A bipolar silver electrode attached to a
Nihon Kohden model SEN-7103 Stimulator (Tokyo, Japan) was used for
nerve stimulation.
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Fig. 1.
Schematic representation of the sites of electrical
stimulation, blood flow measurements, and microinjections of lidocaine
or kainic acid. Stimulation sites: central cut end of the lingual nerve
(LN; A), trigeminal spinal nucleus (Vsp; B), and
inferior salivatory nucleus (ISN; C). Blood flow measurement
site: lower lip [by laser-Doppler flowmeter (LDF)]. Microinjection
sites: Vsp (B) and ISN (C). Dashed lines,
parasympathetic fibers [vasodilator fibers to the lower lip from the
ISN]; solid lines, trigeminal and facial sensory pathways to and
within the brain stem. NTS, nucleus of the solitary tract; OG, otic
ganglion; SSN, superior salivatory nucleus; V, trigeminal nerve root;
VII, facial nerve root; IX, glossopharyngeal nerve root.
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Electrical stimulation of Vsp or the inferior salivatory nucleus.
The animal was mounted in a stereotaxic frame (Narishige, Tokyo,
Japan), and, after a partial craniotomy, part of the tentorium cerebelli was removed by drilling. As indicated schematically in Fig.
1, a guide cannula (1.00 mm OD) was positioned within the Vsp
[posterior (P), 10-11; lateral (L), 5.0-6.0; height (H), 5.0-6.0 mm; coordinates of Berman (3)] or the
inferior salivatory nucleus (ISN) (P, 8-9; L, 2.5-3.5; H,
5.0-6.0 mm) via a small burr hole in the skull. This was achieved
with the aid of a micromanipulator and without removing any part of the
brain. A concentric bipolar electrode (Inter Medical, Tokyo, Japan),
insulated with enamel, except at the tip, was inserted through the
guide cannula. A lower lip vasodilator site in Vsp or ISN was
identified by lowering the stimulating electrode until a maximal
vasodilator response was elicited by electrical stimulation. Quite
small movements of the stimulating electrode in the vertical direction
(i.e., 0.5 mm) often markedly altered the response to stimulation,
indicating that current spread from the electrode tip was not
excessive. For electrical stimulation of Vsp or ISN, we routinely used
a 20-s train of rectangular pulses generated by a Nihon Kohden model SEN-7103 stimulator through an isolation unit (Nihon Kohden model SS-202J), usually with a current of 100 µA and a pulse duration of 2 ms at a frequency of 10 Hz. The sites of electrical stimulation and
those at which microinjections were made were examined histologically, as described below.
Microinjections of lidocaine and kainic acid.
To determine whether the vasodilator response elicited by LN
stimulation was mediated via Vsp or ISN, lidocaine (2%) or kainic acid
(10 mM) was microinjected into Vsp or ISN in a volume of 1.0 µl/site
via an injection cannula (0.50 mm OD) inserted through the previously
implanted guide cannula. The stimulating electrode was interchangeable
with the injection cannula. Both were of equal length and each extended
5.0 mm beyond the tip of the guide cannula. Thus microinjection and
electrical stimulation were carried out at the same sites. Saline (1.0 µl) was used for control injections. It never produced any
significant effect on the LN-evoked vasodilatation or on resting
cardiovascular parameters. The magnitude of the LN-evoked response
obtained after microinjection of a given agent was expressed as a
percentage of the control response recorded before its administration
(mean ± SE).
Measurement of lower lip blood flow and of SABP.
Blood flow changes in the lower lip were monitored (Fig. 1) using a
laser-Doppler flowmeter (LDF; model ALF21D; Advance, Tokyo, Japan), as
described before (13, 16, 22). The probe was placed
against the lower lip without exerting any pressure on the tissue. The
blood flow changes were assessed by measuring the height of the
response on the chart. In Figs. 3, 4, and 6-9, flow levels
are expressed in arbitrary units and % of control.
SABP was recorded from the femoral catheter via a Statham pressure
transducer. A tachograph (model AT-610G; Nihon Kohden) triggered by the
arterial pulse was used to monitor heart rate.
Histology.
Animals were given an overdose of pentobarbital sodium (60 mg/kg)
by intravenous infusion and perfused through the ascending aorta with
1.0-2.0 liters of saline (0.9%) followed immediately by 2 liters
of 10% formaldehyde. Then, the brain stem and upper cervical spinal
cord were removed and stored for 1-4 days in buffered 30%
sucrose. After storage, sections 50-µm thick were cut on a freezing
microtome and collected in 0.1 M phosphate buffer (pH 7.4). Sections
were mounted on gelatin-coated slides and stained with thionin.
Photomicrographs of representative coronal sections showing sites used
for electrical stimulation or microinjection of lidocaine or kainic
acid into the Vsp can be seen in Fig. 2.
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Fig. 2.
Photomicrographs of representative coronal sections through the
medulla oblongata of the cat showing sites at which electrical
stimulation and microinjections of lidocaine or kainic acid were
delivered to the Vsp (A) and ISN (B). Thionin
stain, original magnification ×15. FN, facial nucleus; FTG,
gigantocellular tegmental field; PH, nucleus praepositus hypoglossi;
RB, restiform body; RFN, retrofacial nucleus; VIN, inferior vestibular
nucleus; VLD, lateral vestibular nucleus; VMN, medial vestibular
nucleus; Vt, trigeminal spinal tract.
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Pharmacological agents.
To examine whether the LN- and Vsp-evoked vasodilator responses
were mediated via activation of the autonomic nervous system, hexamethonium, an autonomic ganglion (cholinergic) blocker, was administered (1.0 mg/kg iv) and stimulation was repeated, beginning 10 min later. The magnitude of the responses obtained was expressed as a
percentage of the response elicited by electrical stimulation of the LN
or Vsp before hexamethonium (mean ± SE).
Statistical analysis.
All numerical data are given as means ± SE. The significance
of changes in the test responses was assessed using ANOVA followed by
either a post hoc test (Tukey-Kramer) or a contrast test. Differences were considered significant at the level P < 0.05. Data were analyzed using a Macintosh computer with StatView 5.0 and
Super ANOVA.
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RESULTS |
The resting mean arterial blood pressure of the cats used in this
study lay within the range 74.3 ± 8.7 to 127.1 ± 10.4 mmHg, the average value for the group being 91.9 ± 9.0 mmHg.
Effects of microinjections of lidocaine or kainic acid into Vsp.
Local anesthesia of the ipsilateral Vsp by microinjection of
lidocaine was produced to examine the contribution made by this area to
the LN-evoked vasodilator response. Figure
3A shows a typical recording
of the time course of the effects of such lidocaine microinjection on
the vasodilator response elicited reflexly by LN stimulation. Mean data
(Fig. 4) show that the reflex
parasympathetic vasodilatation was significantly reduced by
microinjection of lidocaine (2%, 1 µl/site) into Vsp
[F(6,24) = 11.263, n = 5, P < 0.001] and that the response recovered in a
time-dependent manner. The microinjection sites are shown in Fig.
5B.
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Fig. 3.
A: typical example of inhibitory effect produced by
microinjection of lidocaine (1 µl/site) into the ipsilateral Vsp on
the blood flow increase in lower lip (LBF) [in arbitrary units (au)]
elicited by electrical stimulation of the central cut end of the
lingual nerve (LN). Electrical stimulation of the LN was at 30 V, 10 Hz, 2-ms pulse duration for 20 s. B: typical example of
inhibitory effect produced by microinjection of kainic acid (1 µl/site) into the right Vsp on the LBF increase (in au) elicited by
electrical stimulation either of the central cut end of LN [right side
(top recordings) or left side (bottom
recordings)] or of the ISN (right side). Kainic acid and lidocaine
(see A) were microinjected at the same site in Vsp.
Electrical stimulation was carried out before and after the
microinjection of kainic acid at 30 V, 10 Hz, 2-ms pulse duration for
20 s (LN) or at 100 µA, 10 Hz, 2-ms pulse duration for 20 s
(ISN). Electrical stimulation of either LN or ISN was carried out
before kainic-acid microinjection into the Vsp, then repeated 60 min
after the kainic acid-induced blood flow increase had returned to the
basal level.
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Fig. 4.
Time course of the effect produced by microinjection of
lidocaine (1 µl/site;  ) or of kainic acid (1 µl/site;  ) into the ipsilateral Vsp on the LBF
increase elicited by stimulation of the central cut end of the LN.
Electrical stimulation of LN was at 30 V, 10 Hz, 2-ms pulse duration
for 20 s. After microinjection of kainic acid, electrical
stimulation of LN was performed only after the kainic acid-induced
blood flow increase had returned to the basal level. Each value is
expressed as a percentage of the pretreatment response (at time
0) and is given as means ± SE. Statistical significance from
control (at time 0) was assessed by means of ANOVA followed
by a contrast test (*P < 0.05, **P < 0.01, ***P < 0.001). Number of animals used is shown in
parentheses.
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Fig. 5.
Location of stimulation sites within the brain stem. A:
sites from which vasodilator responses were induced by electrical
stimulation of the ISN () or Vsp ().
B: sites at which microinjection of lidocaine into ISN
() or of kainic acid and also lidocaine into the Vsp
() attenuated vasodilator responses induced by
electrical stimulation of the lingual nerve. IO, inferior olivary
complex; PH, nucleus praepositus hypoglossi; P, posterior.
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Kainic acid (10 mM/site, 1 µl) was microinjected into Vsp at sites at
which microinjection of lidocaine evoked a marked reduction in the
vasodilator response evoked by stimulation of the ipsilateral LN. This
was done to examine the relative contribution made by cell bodies in
this area to the response (Fig. 3B). Kainic acid was given
20-30 min after the LN-evoked response had completely recovered
from the effects of lidocaine. The microinjection sites are shown in
Fig. 5B. Mean data (Fig. 4) show that the reflex parasympathetic vasodilatation was significantly reduced by
microinjection of kainic acid into Vsp
[F(6,24) = 9.009, n = 5, P < 0.001] and that this effect of kainic acid was
irreversible (at least within the time frame of this experiment).
Microinjection of kainic acid itself into this area evoked a
long-lasting vasodilatation in the ipsilateral lower lip (Fig.
3B). Figure 3B also shows the effects
1) of electrical stimulation of ISN on the side ipsilateral to the recording site in the lower lip and 2) of electrical
stimulation of the contralateral LN. Electrical stimulation of ISN
elicited vasodilatation in the ipsilateral lower lip regardless of
whether the Vsp had been damaged by kainic acid. Kainic acid did not
affect the vasodilatation in the lower lip evoked by stimulation of the contralateral LN (bottom trace in Fig. 3B). The
mean data in Fig. 6 show that kainic-acid
microinjection into Vsp markedly reduced the vasodilatation evoked by
stimulation of the ipsilateral LN (P < 0.001) but had
no effect on that evoked by stimulation of the ipsilateral ISN.
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Fig. 6.
Effects produced by microinjection of kainic acid (1 µl/site) into the Vsp on the LBF increase evoked by electrical
stimulation of the central cut end of the LN or of the ISN (both
ipsilateral to the microinjection site). Electrical stimulation of
either LN or ISN was carried out before kainic acid microinjection into
the Vsp, then repeated 60 min after the kainic acid-induced blood flow
increase had returned to the basal level. Open and hatched bars
indicate, respectively, control responses and responses obtained after
pretreatment with kainic acid. Each value is expressed as a percentage
of the pretreatment response and is given as means ± SE.
Statistical significance of difference from control was assessed by
means of ANOVA followed by a contrast test (P < 0.001).
Number of animals used is shown in parentheses.
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Effects of microinjection of lidocaine into ISN.
Figure 7A shows typical
recordings of the time course of the effects produced by microinjection
of lidocaine into ISN on the lower lip vasodilatation evoked by
electrical stimulation of LN or Vsp. The microinjection sites are shown
in Fig. 5B. Mean data (Fig. 7B) show that
lidocaine microinjection elicited a time-dependent attenuation of both
the LN- and Vsp-induced vasodilatations [for LN:
F(6,24) = 9.364, n = 5, P < 0.001; for Vsp:
F(6,36) = 7.948, n = 7, P < 0.001] and that both showed time-dependent
recovery.
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Fig. 7.
A: typical examples of inhibitory effects produced by
microinjection of lidocaine (1 µl/site) into the ISN on the LBF
increase (in au) elicited by electrical stimulation either of the
central cut end of the LN () or of the Vsp
() (both ipsilateral to the microinjection site).
Electrical stimulation was at 30 V, 10 Hz, 2-ms pulse duration for
20 s (LN) or at 100 µA, 10 Hz, 2-ms pulse duration for 20 s
(Vsp). B: time course of effect produced by microinjection
of lidocaine (1 µl/site) into the ISN on the LBF increase elicited by
stimulation either of the central cut end of LN () or
of Vsp () (both ipsilateral to the microinjection
site). Electrical stimulation was at 30 V, 10 Hz, 2-ms pulse duration
for 20 s (LN) or at 100 µA, 10 Hz, 2-ms pulse duration for
20 s (Vsp). Each value is expressed as a percentage of the
pretreatment response (at time 0) and is given as means ± SE. Statistical significance from control (at time 0) was
assessed by means of ANOVA followed by a contrast test (*P
< 0.05, **P < 0.01, ***P < 0.001). Number
of animals used is shown in parentheses.
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Vasodilator responses to Vsp stimulation.
Figure 8A shows typical
examples of the effects of electrical stimulation of Vsp at various
intensities from 10 to 200 µA on blood flow in the ipsilateral lower
lip and on SABP. Increasing the stimulus intensity from 20 to 200 µA
elicited an intensity-related blood flow increase in the lower lip, but
did not evoke a significant change in SABP (Fig. 8A). Mean
data show that the threshold intensity needed to elicit the vasodilator
response was 20 µA and that the response was saturated by 100 µA
(Fig. 8B). The stimulation sites are shown in Fig.
5A.
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Fig. 8.
A: typical example of dependence on stimulus
intensity shown by LBF changes evoked by electrical stimulation of the
Vsp. Traces show blood flow responses in lower lip (in au) and systemic
arterial blood pressure (SABP; in mmHg). B and C:
stimulus intensity response (B) and frequency response
(C) relationships for LBF changes evoked by electrical
stimulation of Vsp. Stimulation was at various intensities (10-200
µA) and various frequencies (0.1-100 Hz). Intensity response
curve was generated using stimulus trains at 10 Hz. Frequency response
curve was generated using stimulus trains at 100 µA. Each value is
given as means ± SE. Number of animals used is shown in
parentheses. D: changes in SABP (mmHg) evoked by electrical
stimulation of LN, Vsp, or ISN. Number of animals used is shown in
parentheses.
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Figure 8C shows the effect of varying the frequency [from
0.1 to 100 Hz for 20 s at 2-ms pulse duration and 100 µA] of
Vsp stimulation on the vasodilator response in the lower lip. At
frequencies <10 Hz, the vasodilator response increased progressively
with the frequency. At frequencies >10 Hz, no additional increase, rather a decrease, in blood flow was observed. The optimal frequency was therefore 10 Hz.
Figure 8D shows that the changes in SABP were not
consistent whether electrical stimulation was delivered to LN, Vsp, or
ISN. A given animal always produced the same SABP response (i.e., an increase or decrease) regardless of whether stimulation was applied to
LN (group mean ± SE, +1.0 ± 2.7 mmHg, n = 14) or Vsp (
6.8 ± 3.8 mmHg, n = 17). On the
other hand, electrical stimulation of ISN did not evoke an increase in
SABP in any animal, only no change or a decrease being seen (0.9 ± 0.5 mmHg, n = 13). The averaged data (also given
numerically in Fig. 8D) showed no statistically significant
difference among the regions stimulated.
Effects of an autonomic ganglion-blocking agent.
Figure 9A shows typical
recordings of the time course of the effects produced by the autonomic
ganglion-blocking agent hexamethonium (1 mg/kg iv) on the vasodilator
responses elicited by electrical stimulation of LN or Vsp. Mean data
(Fig. 9B) show that hexamethonium reduced both vasodilator
responses [for LN: F(6,18) = 6.404, n = 4, P < 0.001; for Vsp:
F(6,30) = 8.709, n = 6, P < 0.001] and that both showed time-dependent
recovery.
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Fig. 9.
A: typical examples of inhibitory effects of
hexamethonium (1.0 mg/kg iv) on the LBF increase (in au) elicited by
electrical stimulation either of the central cut end of the LN
() or of the Vsp (). Electrical
stimulation was at 30 V, 10 Hz, 2-ms pulse duration for 20 s (LN)
or at 100 µA, 10 Hz, 2-ms pulse duration for 20 s (Vsp).
B: time course of the effect of hexamethonium (1.0 mg/kg iv)
on the LBF increase elicited by electrical stimulation either of the
central cut end of LN () or of Vsp ().
Electrical stimulation was at 30 V, 10 Hz, 2-ms pulse duration for
20 s (LN) or at 100 µA, 10 Hz, 2-ms pulse duration for 20 s
(Vsp). Each value is expressed as a percentage of the pretreatment
response (at time 0) and is given as means ± SE.
Statistical significance from control (at time 0) was
assessed by means of ANOVA followed by a contrast test (*P
< 0.05, **P < 0.01, ***P < 0.001). Number
of animals used is shown in parentheses.
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DISCUSSION |
Some years ago, we proposed the presence of a parasympathetic
reflex vasodilator mechanism serving the orofacial areas in the cat
(19, 21). Although the afferent and efferent pathways involved in this reflex response to somatic sensory stimulation have
now been well studied [see reviews by Izumi (14, 15)], the central mechanism in the brain stem remains uncertain. We now
report evidence suggesting that Vsp is involved in mediating the
LN-evoked parasympathetic reflex vasodilatation in the cat lower lip.
The parasympathetic reflex vasodilatation in the lower lip evoked by
electrical stimulation of the central cut end of LN was reduced by
82.6 ± 8.9% at 10 min after microinjection of lidocaine into Vsp
(Figs. 3A and 4). The reduction lasted nearly 30-40
min. This indicates that either Vsp or vasodilator fibers passing near Vsp are involved in mediating this response. In the present study, 1.0 µl of lidocaine was microinjected into Vsp to inhibit neuronal traffic through this part of the medulla because 1) 1.0 µl
of lidocaine microinjected 2.0 mm either lateral or dorsal to Vsp had
no inhibitory effect on the LN-evoked vasodilatation and 2) electrical stimulation of sites 2.0 mm away from Vsp did not evoke a
parasympathetic vasodilator response in the lower lip. These findings
are in accord with the observation of Gebhart et al. (11)
that 1.0 µl of lidocaine microinjected into the medulla in cats had
no effect on the efficacy with which inhibition of spinal dorsal horn
neurons was produced by electrical stimulation at a site 2.0 mm lateral
to the injection site. In our hands, microinjection of lidocaine into
Vsp produced a reversible inhibition of the LN-evoked parasympathetic
vasodilatation. Although injection of a vasoconstrictor (such as
epinephrine) with the lidocaine would have helped restrict the spread
of the local anesthetic, we did not do this in the present experiment
to avoid the possible effect of the vasoconstrictor itself on the
parasympathetic vasodilator response evoked by LN stimulation via
either activation or inhibition of brain stem neurons. Thus we consider
that microinjection of lidocaine is a more useful way of examining the
possible involvement of specific nuclei in the brain than making
electrolytic lesions, because the latter produce irreversible damage
(nor can the tissue functionally affected be precisely determined by
subsequent histologic evaluation alone, although at first sight this
may appear to be its advantage) (36, 40). However, the
main limitation of the use of lidocaine is that it is impossible to
determine if changes occurring after its microinjection result from
effects on fibers en passage or on neuronal cell bodies residing within
the area.
On the other hand, it is well known that the lesions produced by kainic
acid or ibotenic acid destroy cell bodies without disrupting fibers of
passage (25, 28, 34). Unilateral microinjection of kainic
acid into the ipsilateral Vsp markedly reduced the LN-evoked vasodilatation but had no significant effect on the ISN-evoked vasodilatation (Figs. 3B and 6). Recovery from this
inhibitory effect did not occur within 3 h and thus the effect
seemed to be irreversible. These results indicate that cell bodies
within or near Vsp constitute an essential bulbar relay for the
LN-evoked vasodilatation in the lower lip. Furthermore, because the
response to LN stimulation was not affected by microinjection of kainic acid into the contralateral Vsp, the relay would appear to be involved
in the ipsilateral reflex arc only.
As shown in Fig. 8, electrical stimulation of Vsp elicited
vasodilatation in the lower lip usually without increasing SABP in our
vagosympathectomized cats. There were no statistically significant
differences from baseline SABP at either 10 or 20 min after
microinjection of the GABA agonist muscimol (0.5 mg/kg) into the Vsp.
However, a slight but statistically significant decrease in SABP
(P < 0.05) was observed 30 min after muscimol injection (104.0 ± 7.4, 102.7 ± 7.7, 99.2 ± 6.6, and
95.3 ± 6.4 mmHg at 0, 10, 20, and 30 min after muscimol
administration) [F(3,18) = 7.08, n = 7, P < 0.01 by ANOVA with post hoc
test (Tukey-Kramer) for repeated measurements]. Bousquet et al.
(7) found that microinjections of the same dose of
muscimol into the nucleus of the solitary tract (NTS) in pentobarbital
sodium-anesthetized cats produced hypertension and tachycardia. We,
therefore, feel that the inhibitory effects of lidocaine and kainic
acid reported here are most likely due to effects on structures located
within or very near Vsp, not on NTS [which is situated 2-3.5 mm
lateral to obex, a location clearly different from that of Vsp (at
6.0-7.0 mm anterior, 5.0-6.0 mm lateral, and 1.0-3.0 mm
ventral to obex)]. However, we cannot exclude some involvement by NTS
in the responses under study (see below). The effects produced by the
autonomic ganglion-blocker hexamethonium (Fig. 9) suggest that Vsp
stimulation-evoked vasodilatation is mediated via a parasympathetic
mechanism (because the cervical sympathetics were cut).
We need to consider whether preganglionic parasympathetic fibers
issuing from the superior and inferior salivatory nuclei [located near
the Vsp/trigeminal tract (VT)] or indeed the inferior salivatory
nucleus itself (containing the preganglionic parasympathetic cell
bodies) might have been stimulated (or blocked) when electrical stimulation (or lidocaine or kainic acid) was notionally delivered to
Vsp, because in the cat the facial and glossopharyngeal parasympathetic preganglionic fibers cross Vsp/VT (30, 31) and their cell bodies are located near Vsp/VT (12, 35). One possible
argument against the above scenario is that the sites at which the
preganglionic parasympathetic fibers forming the efferent (as well as
afferent) components of the chorda tympani and glossopharyngeal nerves
cross Vsp/VT are ~3-4 mm anterior to the sites at which
stimulation or microinjection was delivered to Vsp [and neither
microinjected lidocaine (1 µl/site) nor electrical stimulation of Vsp
at 100 µA is likely to have spread that far (see above)]. However,
this does not enable us to exclude spread of kainic acid to
preganglionic parasympathetic cell bodies in or near ISN as a factor in
our results and this must remain a subject for further inquiry.
Although our data strongly suggest that Vsp participates as a relay in
the LN-evoked parasympathetic reflex vasodilator response in the lower
lip, the involvement of the NTS (if any) remains unclear. It has been
reported that both gustatory and trigeminal afferents running in LN
pass to NTS and Vsp [via the chorda tympani, greater superficial
petrosal nerves (VII), lingual-tonsillar nerves (IX) and the superior
laryngeal nerves (X) (5), and the trigeminal primary
(32, 37)]. Those afferents passing to the trigeminal nucleus apparently subserve nociceptive and tactile sensations, whereas
those passing to NTS subserve gustatory sensation. We previously
reported that pinching, electrical stimulation with higher intensities,
topical application of capsaicin, and radiant heat stimulation to the
tongue all caused an increase in the ipsilateral LBF, whereas
nonnociceptive mechanical stimulation did not (20, 24),
and that all of the LBF increases evoked by these stimulations were
significantly attenuated by pretreatment with an autonomic ganglionic blocker, hexamethonium. These results suggest that the
C-polymodal nociceptor is a strong candidate for the primary afferent partaking in the LN stimulation-induced reflex parasympathetic LBF increase. The present results showing a very marked reduction of
such vasodilatation after microinjection of lidocaine or kainic acid
into the Vsp seems to suggest that gustatory afferent stimulation may
not elicit reflex parasympathetic vasodilatation in the lower lip. Put another way, our results suggest a poor involvement of NTS in
this reflex arc. However, it is conceivable that Vsp stimulation excited the parasympathetic vasodilator nucleus via an activation of
NTS. This point is now under investigation in our laboratory. It is
possible that the parasympathetic reflex vasodilatation seen in the
present study requires at least a four-neuron pathway: trigeminal
afferents-Vsp-parasympathetic preganglionic neurons located in the
inferior salivatory nucleus-otic postganglionic neurons. This notion is
supported by an anatomical study showing that the parasympathetic
preganglionic neurons receive projections from Vsp (38).
Finally, the question arises as to which part(s) of Vsp might be
involved in the pathway under study. The rostral components of the
trigeminal spinal nucleus [i.e., the subnucleus oralis (Vo) and
subnucleus interpolaris (Vi)] have been implicated in orofacial
nociceptive mechanisms related especially to intraoral/perioral pain
(8, 9, 33). The caudal component of the trigeminal spinal
nucleus (Vc) has traditionally been viewed as an essential relay site
for nociceptive information from superficial and deep craniofacial
tissues. Furthermore, it has been suggested that Vc contributes to the
control of such autonomic functions as the adrenal secretion of
catecholamines (1, 2, 10). In the present study, the
bulbar sites at which microinjection of lidocaine or kainic acid
attenuated or abolished the LN-evoked reflex vasodilatation were
situated ~6.0-7.0 mm anterior, 5.0-6.0 mm lateral, and
1.0-3.0 mm ventral to obex [atlas of Berman (3)],
an area that seems to correspond to Vi and/or to Vo. On this basis, we
tentatively suggest that Vi and/or Vo might contribute as a relay to
the parasympathetic reflex vasodilator response in the lower lip and
possibly to other autonomic responses.
Perspectives
The nature of the adequate stimulus, the identity of the receptors
for the LN stimulation-induced LBF increase, and the characteristics of
the afferent fibers that mediate it are unknown. Because it can be
evoked from all branches of the trigeminal nerve, it is unlikely to
arise from a specific cranial organ (e.g., the eye, teeth, nasal
mucosa, or tongue), nor can it be related to a specific division of the
trigeminal nerve. This suggests that the parasympathetic reflex
vasodilatation that occurs in response to trigeminal stimulation does
not arise from proprioceptors. The trigeminal system appears to
participate in autonomic functions such as salivation, lacrimation, and
vasomotor and other cardiovascular responses (6, 19, 27,
41). These are independent of the role of the system in somatosensory perception, because they can be observed in the unconscious individual, for example during surgical anesthesia, or even
in a decerebrate animal (6, 17, 27). Our findings that the
Vsp modulates parasympathetic reflex vasodilatation add a new facet to
the notion that the trigeminal system may serve to link the somatic and
autonomic nervous systems.
| |
ACKNOWLEDGEMENTS |
This study was partly supported by a grant-in-aid for scientific
research from the Ministry of Education, Science, Sports and Culture of
Japan [No. 12671797 (H. Izumi) and No. 13672140 (T. Saito)].
| |
FOOTNOTES |
Address for reprint requests and other correspondence: H. Izumi, Dept. of Oral Molecular Bioregulation, Tohoku Univ. School of
Dentistry, Sendai 980-8575, Japan (E-mail:
izumi{at}physiol.dent.tohoku.ac.jp).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 16 July 2001; accepted in final form 16 October 2001.
| |
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ABSTRACT
INTRODUCTION
